System and method for determining the concentration of an analyte in a sample fluid

ABSTRACT

The present disclosure relates to various methods for measuring the amount of an analyte present in a biological fluid using an electrochemical testing process. Various embodiments are disclosed, including the use of AC test signals and the performance of tests having a Total Test Time within about 3.0 seconds or less, and/or having a clinically low Total System Error.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Utility applicationSer. No. 13/418,611, filed Mar. 13, 2012. U.S. patent application Ser.No. 13/418,611 is a divisional application of U.S. Utility patentapplication Ser. No. 12/650,065, filed Dec. 30, 2009, now issued as U.S.Pat. No. 8,148,164, issued Apr. 3, 2012. U.S. Utility patent applicationSer. No. 12/650,065 is a continuation-in-part of U.S. Utility patentapplication Ser. No. 10/871,966, filed Jun. 18, 2004, now issued as U.S.Pat. No. 7,749,437, issued Jul. 6, 2010, which claims the benefit ofU.S. Provisional Application No. 60/480,397, filed Jun. 20, 2003. U.S.Utility patent application Ser. No. 12/650,065 is also acontinuation-in-part of U.S. Utility patent application Ser. No.10/871,673, filed Jun. 18, 2004, now issued as U.S. Pat. No. 7,727,467,issued Jun. 1, 2010, which claims the benefit of U.S. ProvisionalApplication No. 60/480,397, filed Jun. 20, 2003. U.S. Utility patentapplication Ser. No. 12/650,065 is also a continuation-in-part of U.S.Utility patent application Ser. No. 12/505,124, filed Jul. 17, 2009,which is a divisional of U.S. Utility patent application Ser. No.12/330,757, filed Dec. 9, 2008, now issued as U.S. Pat. No. 7,977,112,issued Jul. 12, 2011, which is a continuation of U.S. Utility patentapplication Ser. No. 10/688,561, filed Oct. 17, 2003, now issued as U.S.Pat. No. 7,488,601, issued Feb. 10, 2009, which claims the benefit ofU.S. Provisional Application No. 60/480,298, filed Jun. 20, 2003. U.S.Utility patent application Ser. No. 12/650,065 is also acontinuation-in-part of U.S. Utility patent application Ser. No.11/746,465, filed May 9, 2007, which is a continuation-in-part of U.S.patent application Ser. No. 10/688,312, now issued as U.S. Pat. No.7,390,667, issued Jun. 24, 2008. The contents of these applications andpatents are hereby incorporated by reference in their entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a measurement method and apparatus foruse in measuring concentrations of an analyte in a fluid. The inventionrelates more particularly, but not exclusively, to a method andapparatus which may be used for measuring the concentration of glucosein blood.

BACKGROUND OF THE INVENTION

Measuring the concentration of substances, particularly in the presenceof other, confounding substances, is important in many fields, andespecially in medical diagnosis. For example, the measurement of glucosein body fluids, such as blood, is crucial to the effective treatment ofdiabetes.

Diabetic therapy typically involves two types of insulin treatment:basal and bolus. Basal insulin refers to continuous, e.g. time-releasedinsulin. Bolus insulin treatment provides additional doses of fasteracting insulin to regulate fluctuations in blood glucose caused by avariety of factors, including the meal-time metabolization of sugars andcarbohydrates, etc. Proper regulation of blood glucose fluctuationsrequires accurate measurement of the concentration of glucose in theblood. Failure to do so can produce extreme complications, includingblindness or impaired circulation in the extremities, which canultimately deprive the diabetic of use of his or her fingers, hands,feet, etc.

Multiple methods are known for measuring the concentration of analytesin a blood sample, such as, for example, glucose. Such methods typicallyfall into one of two categories: optical methods and electrochemicalmethods. Optical methods generally involve reflectance or absorbancespectroscopy to observe the spectrum shift in a reagent. Such shifts arecaused by a chemical reaction that produces a color change indicative ofthe concentration of the analyte. Electrochemical methods generallyinvolve, alternatively, amperometric or coulometric responses indicativeof the concentration of the analyte. See, for example, U.S. Pat. Nos.4,233,029 to Columbus, 4,225,410 to Pace, 4,323,536 to Columbus,4,008,448 to Muggli, 4,654,197 to Lilja et al., 5,108,564 to Szuminskyet al., 5,120,420 to Nankai et al., 5,128,015 to Szuminsky et al.,5,243,516 to White, 5,437,999 to Diebold et al., 5,288,636 to Pollmannet al., 5,628,890 to Carter et al., 5,682,884 to Hill et al., 5,727,548to Hill et al., 5,997,817 to Crismore et al., 6,004,441 to Fujiwara etal., 4,919,770 to Priedel, et al., 6,054,039 to Shieh, and 6,645,368 toBeaty et al., which are hereby incorporated by reference in theirentireties.

For the convenience of the user, reducing the time required to displayan indication of the glucose level in a blood sample has been a goal ofsystem designers for many years. Test times have been reduced from earlycolorimetric products that took approximately two minutes to display areading, to test times on the order of 20-40 seconds. More recently,test times shorter than ten seconds have been described (see, forexample, U.S. Pat. Nos. 7,276,146 and 7,276,147), and several productscurrently on the market advertise test times of about five seconds.Shorter test times of less than two seconds have been discussed invarious patent applications (see, for example, U.S. Patent ApplicationPublication Nos. 2003/0116447A1 and 2004/0031682A1). But the trueutility of a short test time is not completely reached with theseteachings in terms of the results being substantially unaffected byconfounding interferents.

An important limitation of electrochemical methods for measuring theconcentration of a chemical in blood is the effect of confoundingvariables on the diffusion of analyte and the various active ingredientsof the reagent. Examples of limitations to the accuracy of blood glucosemeasurements include variations in blood composition or state (otherthan the aspect being measured). For example, variations in hematocrit(concentration of red blood cells), or in the concentration of otherchemicals in the blood, can effect the signal generation of a bloodsample. Variations in the temperature of the blood samples is yetanother example of a confounding variable in measuring blood chemistry.The utility of a reported blood glucose response after a short test timeis questionable in applications where the results are not compensatedfor other sample variables or interferents such as hematocrit andtemperature.

With respect to hematocrit in blood samples, prior art methods haverelied upon the separation of the red blood cells from the plasma in thesample, by means of glass fiber filters or with reagent films thatcontain pore-formers that allow only plasma to enter the films, forexample. Separation of red blood cells with a glass fiber filterincreases the size of the blood sample required for the measurement,which is contrary to test meter customer expectations. Porous films areonly partially effective in reducing the hematocrit effect, and must beused in combination with increased delay time and/or AC measurements(see below) to achieve the desired accuracy.

Prior art methods have also attempted to reduce or eliminate hematocritinterference by using DC measurements that include longer incubationtime of the sample upon the test strip reagent, thereby reducing themagnitude of the effect of sample hematocrit on the measured glucosevalues. Such methods also suffer from greatly increased test times.

Other attempts to reduce or eliminate hematocrit and temperatureinterference are taught in U.S. Pat. No. 7,407,811, as well as in thedisclosures of the parent cases to this application, in which an ACpotential of a low amplitude is applied to a sample in order todetermine certain sample characteristics based on phase angle (alsoreferred to herein as “phase”) and admittance information from thecurrent response to the AC excitation signal. As it is taught, multiplefrequencies of an AC excitation signal are applied in sequential blocks,followed by a conventional DC excitation signal. However, thosedisclosures indicate the inventors' belief that there are limits to theminimum time each frequency must be applied in order to obtain useful,consistent and reasonably reproducible information, from both the AC andDC excitation signals. Even then, the shortest total test timepractically achievable from a complete AC method was 3 seconds.Alternatively, to achieve a practical analysis in less than 3 seconds, alimit was placed on the number of frequency blocks used during the ACexcitation, i.e. 2 blocks rather than 4. However, reducing the number offrequency blocks used may have a negative affect on the level ofaccuracy attainable in correcting for multiple interferents, e.g.,hematocrit and temperature. As has been taught in these previousdisclosures of AC excitation, correction of the indicated glucose can beachieved for multiple interferents by obtaining multiple correctionfactors, such as the phase and/or admittance response data resultingfrom multiple frequencies of an AC signal excitation. Multiplecorrection factors are particularly beneficial when they measureindividual or different aspects of interferents or when they areinfluenced by one interferent more than the other.

Furthermore, the correction factors or even the measurements used fordetermining the desired analyte concentration may also be used forcalculating and optionally reporting additional parameters such as thehematocrit level or hematocrit range of the blood. By reducing thenumber of potential correction factors, e.g. measuring the phase and/oradmittance from only two rather than three, four or more frequencies ofan AC excitation, potentially useful information could be forsaken.Information such as hematocrit level or hematocrit range, for example,could be useful information for a user, especially for health careproviders in a clinical setting where patients who are more susceptibleto medically significant abnormal hematocrits due to illness ortreatment could be identified during a routine blood glucose test.Providing a hematocrit level, for example, in addition to the glucoseconcentration would be a valuable piece of information in some settings,which could be lost as a result of the solutions presented by the priorart.

Thus, a system and method are needed that more accurately measure bloodglucose, even in the presence of confounding variables, includingvariations in hematocrit, temperature and the concentrations of otherchemicals in the blood. Further needed are such system and method withtest times of less than 2 seconds. A system and method are likewiseneeded that accurately measure any medically significant component ofany biological fluid with test times of less than 2 seconds. It is anobject of the present invention to provide such a system and method.

SUMMARY OF THE DISCLOSED EMBODIMENTS

In one embodiment, a method for determining a concentration of amedically significant component of a biological fluid is disclosed,comprising the steps of: applying a first signal having an AC componentto the biological fluid; measuring a first current response to the firstsignal; applying a second signal comprising a DC signal to thebiological fluid; measuring a second current response to the secondsignal; combining the first and second responses; and determining anindication of the concentration of the medically significant component.In other embodiments, the time for completing the steps is no more thanabout 2 seconds. In yet other embodiments, the Total System Error fromthe method is no more than about 10%. In yet other embodiments, thefirst signal comprises an AC signal comprising a multi-frequencyexcitation waveform wherein different AC frequencies are generallysimultaneously applied rather than sequentially applied in order tominimize the time for completing application of the first and secondsignals.

Other embodiments of a system and method will be understood from thedescription herein and as set forth in the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further described, by way of example only, withreference to the accompanying drawings, in which:

FIG. 1 is a plot of current versus time for measurements using abiosensor having a reagent layer thickness of about 3.6 μm,parameterized for time between sample application and application of theDC excitation.

FIG. 2 is a table showing the glucose, hematocrit and temperature levelsused in a first covariate study described herein.

FIG. 3 tabularly illustrates the excitation signal profile and timingfor a first study described herein.

FIG. 4 graphically illustrates the excitation signal profile and timingfor a first study described herein.

FIG. 5 is a graph of normalized error versus reference glucose foruncorrected measurement data from a first study described herein.

FIG. 6 is a graph of normalized error versus reference glucose for thedata of FIG. 5 corrected using the methods described herein.

FIG. 7 is a plot of current versus time for measurements using abiosensor having a reagent layer thickness of about 1.6 μm,parameterized for time between sample application and application of theDC excitation.

FIG. 8 is a plot of admittance versus time showing stabilization of theAC response in a first study described herein.

FIG. 9 is a plot showing the cross-sectional thickness of the biosensorreagent stripe in a first study described herein.

FIG. 10 is a table showing the glucose, hematocrit and temperaturelevels of whole blood samples used in a first study described herein.

FIG. 11 illustrates the excitation signal profile and timing used for asecond study described herein.

FIG. 12 is a table showing the measurement performance of three reagentthicknesses used in a second study described herein.

FIG. 13 is a plot of normalized error versus reference glucose level fora second study described herein.

FIG. 14 is a Clark Error Grid showing predicted glucose versus referenceglucose for the uncorrected DC data obtained in a second study describedherein.

FIG. 15 is a Clark Error Grid showing predicted glucose versus referenceglucose for the DC data of FIG. 14 corrected using AC measurement data.

FIG. 16 is a plot of one embodiment multi-sine excitation waveform usedin a third study described herein.

FIG. 17A is a table of 200 ms admittance and phase response data for athird covariate blood glucose measurement study obtained using themethods disclosed herein.

FIG. 17B is a graph of admittance magnitude versus hematocrit from thedata table of FIG. 17A.

FIG. 17C is a graph of phase versus hematocrit from the data table ofFIG. 17A.

FIG. 18 is a table showing both uncorrected blood glucose measurementevaluations at several test times, as well as measurement evaluationsfor the same data corrected using the methods disclosed herein, in athird study described herein.

FIG. 19 is a graph of normalized error versus reference glucose foruncorrected measurement data from a third study described herein.

FIG. 20 is a graph of normalized error versus reference glucose for thedata of FIG. 19 corrected using the methods disclosed herein.

FIG. 21 is a Clark Error Grid showing predicted glucose versus referenceglucose for both the uncorrected data of FIG. 19 and the corrected dataof FIG. 20.

FIG. 22 is a table showing both uncorrected blood glucose measurementevaluations at several test times, as well as measurement evaluationsfor the same data corrected using the methods disclosed herein, in afourth study described herein.

FIG. 23 is a graph of normalized error versus reference glucose foruncorrected measurement data from a fourth study described herein.

FIG. 24 is a graph of normalized error versus reference glucose for thedata of FIG. 23 corrected using the methods disclosed herein.

FIG. 25 is a Clark Error Grid showing predicted glucose versus referenceglucose for both the uncorrected data of FIG. 23 and the corrected dataof FIG. 24.

FIG. 26 is a table showing target versus actual values for the resultsof the fourth covariate study described herein.

FIG. 27 is a graph of admittance magnitude versus hematocrit from thefourth covariate study described herein.

FIG. 28 is a graph of phase versus hematocrit from the fourth covariatestudy described herein.

FIG. 29 is an exemplary current response resulting from a measurementsequence comprising a multi-frequency AC excitation waveform followed bya DC excitation, performed on a whole blood sample having a targetglucose concentration of 93 mg/dL and 70% hematocrit.

FIG. 30 is a table of estimated and measured dry coating film thicknessaccording to coat weight in a known wet reagent application process.

FIG. 31 is a graph of admittance magnitude versus hematocrit from thefifth covariate study described herein.

FIG. 32 is a graph of DC current response measured at different testtimes and co-varied by hematocrit.

FIG. 33. is a graph of normalized error versus reference glucose foruncorrected DC measurement data measured at 900 ms, from the fifth studydescribed herein.

FIG. 34 is a graph of normalized error versus reference glucose for DCmeasurement data measured at 900 ms and corrected according to the fifthstudy described herein.

FIG. 35 is a graph of normalized error versus reference glucose for DCmeasurement data measured at 1100 ms and corrected according to thefifth study described herein.

FIG. 36 is a graph of normalized error versus reference glucose for DCmeasurement data measured at 1500 ms and corrected according to thefifth study described herein.

FIG. 37 is a graph of normalized error versus reference glucose for DCmeasurement data measured at 2000 ms and corrected according to thefifth study described herein.

FIG. 38 is a graph of normalized error versus reference glucose for DCmeasurement data measured at 2500 ms and corrected according to thefifth study described herein.

FIG. 39 is a graph of normalized error versus reference glucose for DCmeasurement data measured at 3000 ms and corrected according to thefifth study described herein.

FIG. 40 is a table showing TSE for corrected responses at the differentDC test times corrected according to the fifth study described herein.

FIG. 41 is a Clark Error Grid showing predicted glucose versus referenceglucose for the uncorrected DC response data at 900 ms according to thefifth study described herein.

FIG. 42 is a Clark Error Grid showing predicted glucose versus referenceglucose for the DC response data at 900 ms corrected by response datafrom one AC frequency, according to the fifth study described herein.

FIG. 43 is a Clark Error Grid showing predicted glucose versus referenceglucose for the DC response data at 900 ms corrected by response datafrom another AC frequency, according to the fifth study describedherein.

FIG. 44 is a Clark Error Grid showing predicted glucose versus referenceglucose for the DC response data at 900 ms corrected by response datafrom two AC frequencies, according to the fifth study described herein.

DETAILED DESCRIPTION OF THE VARIOUS EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings, and specific language will be used to describe thoseembodiments. It will nevertheless be understood that no limitation ofthe scope of the invention is intended. Alterations and modifications inthe illustrated device, and further applications of the principles ofthe invention as illustrated therein, as would normally occur to oneskilled in the art to which the invention relates are contemplated andare desired to be protected. In particular, although the invention isdiscussed in terms of a blood glucose test device and measurementmethods, it is contemplated that the invention can be used with devicesfor measuring other analytes and other sample types. Such alternativeembodiments require certain adaptations to the embodiments discussedherein that would be obvious to those skilled in the art.

A system and method according to the present invention permit theaccurate measurement of an analyte in a fluid in an ultra-fast testtime, namely no more than about 2 seconds. In particular, themeasurement of the analyte remains accurate despite the presence ofinterferents, which would otherwise cause error. For example, a bloodglucose meter according to the present invention measures theconcentration of blood glucose within whole blood samples without errorthat is typically caused by variations in the hematocrit level of thesample and the temperature of the sample. The accurate measurement ofblood glucose is invaluable to the prevention of blindness, loss ofcirculation, and other complications of inadequate regulation of bloodglucose in diabetics. An additional advantage of a system and methodaccording to the present invention is that measurements can be made muchmore rapidly and with much smaller sample volumes, making it moreconvenient for the diabetic person to measure their blood glucose.Likewise, accurate and rapid measurement of other analytes in blood,urine, or other biological fluids provides for improved diagnosis andtreatment of a wide range of medical conditions.

It will be appreciated that electrochemical blood glucose meterstypically (but not always) measure the electrochemical response of ablood sample in the presence of a reagent. The reagent reacts with theglucose to produce charge carriers that are not otherwise present inblood. Consequently, the electrochemical response of the blood in thepresence of a given signal is intended to be primarily dependent uponthe concentration of blood glucose. Secondarily, however, theelectrochemical response of the blood to a given signal may be dependentupon other factors, including hematocrit and temperature. See, forexample, U.S. Pat. Nos. 5,243,516; 5,288,636; 5,352,351; 5,385,846; and5,508,171, which discuss the confounding effects of hematocrit on themeasurement of blood glucose, and which are hereby incorporated byreference in their entireties. In addition, certain other chemicals caninfluence the transfer of charge carriers through a blood sample,including, for example, uric acid, bilirubin, and oxygen, therebycausing error in the measurement of glucose.

The various embodiments disclosed herein relate to systems and methodsthat allow shorter test times to be achieved, while still delivering ananalyte measurement (be it blood glucose or another fluid sampleanalyte) corrected for confounding interferents (be they hematocrit andtemperature, or other interferents). Test times of less than twoseconds, including times less than one second, are enabled by thesystems and methods disclosed herein. As used herein, “Total Test Time”is defined as the length of time from sample detection (or sample dosesufficiency, if both are detected) when a first electrical signal is tobe applied to the sample, to the taking of the last measurement used inthe concentration determination calculations.

In addition to shorter Total Test Times, the embodiments disclosedherein result in analyte measurements having lower Total System Error,or “TSE”. TSE generally comprises a combined measure of accuracy andprecision of a system or method. It is typically calculated as (AbsoluteBias)+2*(Precision), where Bias=Average of Normalized Error;Precision=StdDev(Normalized Error). Normalized Error is typicallycalculated relative to a standard reference value. For example, in thecontext of a blood glucose measurement, Normalized Error=(Predictedglucose−Reference glucose) for a Reference glucose sample less than orequal to 75 mg/dl; but Normalized Error=(Predicted glucose−Referenceglucose)*100/(Reference glucose) for a Reference glucose sample greaterthan 75 mg/dl.

As used herein, the phrase “a signal having an AC component” refers to asignal which has some alternating potential (voltage) portions. Forexample, the signal may be an “AC signal” having 100% alternatingpotential (voltage) and no DC portions; the signal may have AC and DCportions separated in time; or the signal may be AC with a DC offset (ACand DC signals superimposed). In the latter instance, the signal maystill be described as having an AC component even though the polarity ofthe variable potential does not alternate.

Examples 1 and 2 describe details of experiments in which Total TestTime was reduced. In each example, an AC block is used in order togenerate correction data to be combined algorithmically with a DCmeasurement, similar to the known measurement sequence utilized in theACCU-CHEK® Aviva meter. That is, multiple AC potential frequencies areapplied in a sequential fashion with current response and othermeasurement data determined for each frequency. However, in Examples 1and 2, the Total Test Time is reduced by reducing the time for eachsequential AC frequency block, as well as the time for the DC block.Example 1 details an experiment using these condensed time blocks in acovariate study, using a biosensor with a common reagent layerthickness. Example 2 details an experiment with the condensed times in acovariate study using biosensors having variable reagent layerthicknesses.

Measurement sequences for Examples 1 and 2 were conducted using anin-house data acquisition test stand (DATS potentiostat) comprising abank of blood glucose meters configured as a multi-meter test standusing a modified code key to program desired measurement parameters.Although the meters could be programmed or configured with a variety ofmethods and durations for a test sequence, there were a few limitations,such as the choice of frequencies available pre-programmed in the meterhardware. This in-house test stand will be hereinafter referred to asthe “DATS”.

Certain embodiments of the present invention disclosed herein generallyutilize the collection of AC test data at multiple frequencies over ashorter time period by using multi-frequency excitation waveformtechniques. Examples 3 and 4 describe the details of experiments inwhich multi-frequency excitation waveforms were used. Thesemulti-frequency excitation waveforms are formed by adding a plurality ofindividual waveforms of varying frequency together so that the fluidsample is excited by multiple frequencies at the same time.Multi-frequency excitation waveforms allow not only short measurementtimes, but also adaptive measurement sequences, because AC signal datacollection does not permanently alter the sensed chemistry in the waythat a DC measurement does because of the alternating polarity of theapplied excitation. Moreover, the additional frequencies of the ACsignals are applied at low excitation AC potentials, per the methodsdisclosed in co-pending published U.S. patent applicationsUS-2004-0157339-A1, US-2004-0157337-A1, 2004/0157338-A1,US-2004-0260511-A1, US-2004-0256248-A1 and US-2004-0259180-A1, in orderto generate a non-faradaic current response from which a phase angleprovides an indication of certain interfering factors, from whichindication a determination of one or more interferent corrections can bemade and used for more accurately determining the analyte concentrationin the fluid sample.

The resulting sample response can then be measured and the contributionfrom each excitation frequency component can be deduced by use ofFourier Transform techniques, such as a Discrete Fourier Transform(DFT). Although the various examples disclosed herein utilize multi-sineexcitation waveforms, those skilled in the art will recognize that themulti-frequency waveform may be constructed using individual waveformshaving any desired shape, such as triangular, square, sawtooth, delta,etc., just to name a few non-limiting examples. The component ACwaveforms used to create the multi-frequency waveform may each have anydesired frequency and any desired amplitude. The use of multi-frequencytechniques not only shortens the time necessary to collect the desireddata (since the AC measurements are made simultaneously rather thansequentially), but also correlates better for correction since thesample is varying less during the data collection corresponding to eachapplied frequency. Also, the AC measurement can be made closer in timeto the DC measurement. Better correlation between the state of thesample during the respective AC and DC measurements allows for betterinterferent compensation even if the sample is not in steady state.

Measurements for Examples 3 and 4 were conducted with an electrochemicaltest stand constructed on the basis of VXI components from Agilent, andprogrammable to apply AC and DC potentials to sensors in requestedcombinations and sequences and to measure the resulting currentresponses of the sensors. Data were transferred from the electrochemicalanalyzer to a desktop computer for analysis using Microsoft® Excel®. Themeasurements could be carried out by any commercially availableprogrammable potentiostat with an appropriate frequency responseanalyzer and digital signal acquisition system. For commercial use, themethod can be carried out in a dedicated low-cost hand-held measurementdevice, such as the ACCU-CHEK® AVIVA™ blood glucose meter, in which thefirmware is configured to enable application of AC signals in amulti-frequency waveform. In such a case the measurement parameters maybe contained in or provided to the firmware of the meter, and themeasurement sequence and data evaluation executed automatically with nouser interaction. For example, using a programmable potentiostat asdescribed above, measurements were conducted and results analyzed in amanner such that Total Test Times of less than 2 seconds after theanalyte-containing sample was applied to a biosensor and detected by theequipment are possible. Similarly, the firmware of the ACCU-CHEK® AVIVA™blood glucose meter may be provided with measurement parametersconfigured and arranged to cause the measurement sequence to occurwithin the same time periods, namely Total Test Times of less than 2seconds after the analyte-containing sample is applied to a biosensorand detected by the meter. The measurement result may be displayed onthe digital display of the meter when the evaluation of the measurementdata is complete, typically 25-50 ms after the last measurement istaken.

Example 1 Sequential Multiple AC Frequency Test with Fast Total TestTime

U.S. Pat. No. 7,407,811 teaches use of sequentially applied multiplefrequency AC blocks followed by a DC block. For example, Example 5described in U.S. Pat. No. 7,407,811 utilizes sequential applications ofAC excitation followed by a DC excitation. The excitation signalcomprised a 10 kHz AC signal applied for approximately 1.8 seconds, a 20kHz AC signal applied for approximately 0.2 seconds, a 2 kHz AC signalapplied for approximately 0.2 seconds, a 1 kHz AC signal applied forapproximately 0.2 seconds, and a DC signal applied for approximately 0.5seconds. The Total Test Time was 3.0 seconds.

In Example 6 of that same patent, it was desired to obtain Total TestTimes as low as 1.1 seconds using the same test strip design that wasused for Example 5 in that patent. In order to achieve this, theinventors did not believe that they could simply apply the sequentialexcitations of Example 5 for shorter periods of time. As stated in thepatent:

-   -   “Using the same test strip 1700 and reagent described above for        Example 5, the excitation profile illustrated in FIG. 24 was        utilized in order to decrease the Total Test Time. As described        above with respect to Example 5, it was determined that the        phase angle at 20 kHz and at 10 kHz were most closely correlated        with the hematocrit estimation. It was therefore decided to        limit the AC portion of the excitation to these two frequencies        in Example 6 in order to decrease the Total Test Time. In order        to make further reductions in Total Test Time, the 10 kHz AC        excitation was applied simultaneously with the DC signal (i.e.        an AC signal with a DC offset), the theory being that this        combined mode would allow for the collection of simultaneous        results for DC current, AC phase and AC admittance, providing        the fastest possible results. Therefore, the 20 kHz signal was        applied for 0.9 seconds. Thereafter, the 10 kHz and DC signals        were applied simultaneously for 1.0 second after a 0.1 second        interval.”        (U.S. Pat. No. 7,407,811, col. 23, 11. 23-40). The inventors of        U.S. Pat. No. 7,407,811 therefore believed that in order to        shorten the Total Test Time below 3.0 seconds, they needed to        remove two of the AC excitation blocks (those at 2 kHz and 1        kHz) and apply one of the remaining two AC excitation blocks        concurrently with the DC excitation.

One reason for this belief in the prior art is illustrated in FIGS. 1and 7, where the measured DC response of a sample applied to the reagentchemistry is shown for various tests where the timing of the applicationof the DC excitation signal after sample application is varied. It canbe seen that when the DC excitation is applied very quickly after sampleapplication, the response does not exhibit the expected Cottrelliandecay, thereby making accurate determinations of the sample glucoseconcentration impossible for fast test times. This is because enzyme andmediator availability, hydration, and diffusion within the reagent layerlimit how soon the DC measurement can be made in a reproducible fashion.Reagent hydration and coating uniformity from sensor to sensor is asignificant factor in how fast the DC response can be measured.

We have found that shorter AC times are possible because information tocorrelate with interferences such as hematocrit is presented in the ACresponse data even at early times. Although there is some stabilizationof AC over the first few 100 ms, the signals for AC even at short timescorrelate well with the hematocrit interference. Using information forall desired frequencies gathered at the same interval from the desiredglucose DC response enables good correlation or correction of the DCglucose response with the AC measured hematocrit interference.

The present Example 1 was conducted to demonstrate the feasibility ofrunning the prior art sequential multiple AC frequency test methodologyat a faster rate using sensors slot die coated with a uniform reagentformulation containing glucose oxidase. (Slot die coating of uniformreagents are described in U.S. Patent Application Publication No.2005/0008537, which is incorporated herein by reference in itsentirety). The sensor electrodes were made by the process of goldsputtering (˜50 ηm) onto Melinex 329 followed by laser ablation througha chrome-on-quartz mask to form the pattern of the conductive layer todefine the working and dose sufficiency electrodes. The structures usedwere similar to that shown in U.S. Pat. No. 7,407,811, at FIG. 33. Theelectrodes included a pair of dose sufficiency electrodes independent ofa pair of measurement electrodes. Measurements were made using the DATS.A benefit of the DATS configuration is the ease of setup and fast,multi-channel data collection in environmental chambers at differenttemperatures. Using a DATS comprising existing meters configured for usewith AC excitation measurement methods did present some limitations interms of programmability including specific transition times requiredbetween blocks and also the available AC frequencies, essentially allbasic, non-programmable aspects of the meters used with the DATS.However, using such existing meters was useful in that the sequentialmultiple AC frequency method examined in Example 1 (and in Example 2,below) could be backwardly compatible for use with the existing meters.

A covariate study was performed with whole blood samples having sevendifferent glucose target concentrations (50, 100, 150, 250, 300, 450 and550 mg/dL), three different hematocrit target concentrations (25%, 45%and 70%) and five different temperatures (8, 14, 24, 35 and 42 degreesC.). The table of FIG. 2 details the whole blood sample compositionsused for this Example 1.

AC data was collected using sequentially applied AC excitation signalsof 10 kHz, 20 kHz, 2 kHz and 1 kHz at 9 mV RMS. Then, after a 100 msopen circuit, a DC potential of 450 mV was applied starting at 1300 ms.DC measurement data was collected every 100 ms starting at 1400 ms, andthe 1525 ms DC data point was analyzed in this Example 1 (i.e. the testsutilized a Total Test Time of 1.525 seconds). (DC data points were takenat times later than the Total Test Time in order to confirm theviability of the shorter Total Test Time. Because the viability of aTotal Test Time at, e.g., 1.525 seconds or less was confirmed, the DCdata points at longer times were not used for calculating finalresults.) The table of FIG. 3 tabulates the excitation signalcomposition and timing, while this data is presented graphically in ageneral way in FIG. 4.

FIG. 5 plots the normalized error versus the reference glucose forapproximately 1600 data points for all 105 covariant samples ([G], %HCT, ° C.), using only the uncorrected DC measurement taken at 1525 ms.Determining the predicted glucose from the DC measurement was performedusing well known prior art techniques. As will be readily apparent tothose skilled in the art, the performance of the measurement system withsuch a short DC-only test time is extremely poor, with a Total SystemError of 51%.

As shown in FIGS. 3 and 4, the AC excitation potentials (1 through 5)for this Example 1 were applied sequentially. The sequence described wasstarted after a 10 kHz signal was first applied (not shown) to detectsample application (dose detection) and the filling of the capillarytest chamber (sample sufficiency) determination. The use of ACmeasurements for drop detect and dose sufficiency is described in U.S.Pat. No. 7,597,793, which is hereby incorporated herein by reference.

After the sample sufficiency determination, a 300 ms 10 kHz block wasapplied for AC stabilization, followed by four additional AC data blockseach of 100 ms duration at 10 kHz, 20 kHz, 2 kHz and 1 kHz signals. Alltimes herein are started in relation to detection of sample dosesufficiency. Furthermore, Block 1 was kept in the sequence of FIG. 3mainly for backward compatibility to an ACCU-CHEK® AVIVA™-related meterfailsafe, which is not relevant to the present invention. In addition,Block 1 was used to stabilize the AC prior to the next block at the samefrequency. One of the goals of some of this work was to show a backwardscompatible short test time for the product platform of ACCU-CHEK® AVIVA™meters. Additional experiments were conducted with only two frequenciesto examine the limits of the effectiveness of correction in sequentialAC/DC vs. test time. See, e.g. FIG. 8.

After the AC measurements, the measurement electrodes were then held atan open circuit for 100 ms, followed by application of a 450 mV DCsignal. Between each excitation block, there was a 75 ms delayconsisting of a 50 ms pre-stabilization and a 25 ms trailing datacommunication period. Test times were evaluated at 1525 ms (TestTime=1.525 seconds uses DC at 1.5 sec+0.025 s communication time)

AC admittance values were captured for each of the four 100 ms ACexcitation blocks in order to correct the DC glucose measurement for theinterfering effects of hematocrit and temperature using the followingequation:

PredictedGlucose=INT+Yi2*Y2+Pi2*P2+Yi3*Y3+Pi3*P3+Yi4*Y4+Pi4*P4+Yi5*Y5+Pi5*P5+exp(SLOPE+Ys2*Y2+Ps2*P2+Ys3*Y3+Ps3*P3+Ys4*Y4+Ps4*P4+Ys5*Y5+Ps5*P5)*DC**POWER  (equation1)

where: Yi2, Yi3, Yi4, Yi5, Ys2, Ys3, Ys4 and Ys5 are constants

Pi2, Pi3, Pi4, Pi5, Ps2, Ps3, Ps4 and Ps5 are constants

Y2 is the admittance magnitude at 10 kHz (second block)

Y3 is the admittance magnitude at 20 kHz

Y4 is the admittance magnitude at 2 kHz

Y5 is the admittance magnitude at 1 kHz

P2 is the phase angle at 10 kHz (second block)

P3 is the phase angle at 20 kHz

P4 is the phase angle at 2 kHz

P5 is the phase angle at 1 kHz

INT is the intercept

SLOPE is the slope

DC is the uncorrected glucose response predicted with the DC measurement

POWER=Const+Yp2*Y2+Pp2*P2+Yp3*Y3+Pp3*P3+Yp4*Y4+Pp4*P4+Yp5*Y5+Pp5*P5

Equation 1 demonstrates that the system's dose-response can beapproximated by a power model. The slope and power of this power modelare influenced by covariates such as temperature and hematocrit. Sincethe AC measurements (admittance and phase) are sensitive to thesecovariates, they are used in the slope and power terms to compensate forthe covariate effects. The parameter estimates are established byparameter estimation with data collected where glucose, temperature andhematocrit are covaried. The DC value in this example was selected fromone measured DC point, and Equation 1 is specific to a single DC value.For more DC values, i.e. more than one current response measurementtaken during the DC block, a more general representation is:

PredictedGlucose=ba0+a1*Ieff+a2*Peff+a3*Yeff+(b4+exp(b0+b2*Peff+b3*Yeff))*Ieff**(c0+c2*Peff+c3*Yeff)

where: Ieff=bV0+bV1*DC1+bV2*DC2+bV3*DC3+bV4*D4+bV5*DC5+bV6*DC6

Peff=bP0+bP1*P1+bP2*P2+bP3*P3+bP4*P4+bP5*P5+bP6*P6

Yeff=bY0+bY1*Y1+bY2*Y2+bY3*Y3+bY4*Y4+bY5*Y5+bY6*Y6

The use of AC admittance magnitude and phase data to correct DC glucoseresponse data for the effects of hematocrit and temperature is discussedin detail in U.S. Pat. No. 7,407,811.

Like FIG. 5, FIG. 6 also plots the normalized error versus the referenceglucose for all 105 samples, except that the DC measurement taken at1525 ms has been corrected using the AC measurements and the methodologydiscussed hereinabove. Such correction allows the measurement system tocompensate for the interfering effects of hematocrit and temperature. Ascan be seen, all measurement results now fall with +/−15% normalizederror, with a total system error of 9.4%, all with a test time of only1.525 seconds.

This Example 1 therefore demonstrates that an extremely short test timeof 1.525 seconds can be achieved using multiple serial AC excitationfrequencies in order to probe the sample and measure interferents thatprevent accurate assessment of the glucose value, and to correct themeasured glucose value to remove the effects of these interferents uponthe measurement. This surprising result is in contravention to theteachings of the prior art as pointed out above.

Control of Reagent Thickness

Some of the embodiments disclosed herein, including embodiments shownfrom the description of Example 2 below, also utilize accurate controlof the biosensor reagent thickness through the use of a uniform methodof applying the reagent to the biosensor surface, such as by slot diecoating, for example. The reagent coatings disclosed herein aregenerally about 1.6-5 μm in thickness. The uniformity of the reagentcoating, and thus the resulting uniform dissolution/hydration of thereagent film with the fluid sample, enables reproducibility thatcorrelates well with the AC measurements to provide accuratelycompensated glucose. Non-uniform reagent thicknesses are detrimental toachieving faster methods and improved performance because of morevariability in the measurements, especially at short times. For robustperformance, we strive for a very uniform film. For a description ofmethods and disclosure relating to coating uniform films, see U.S.Patent Application Publication No. 2005/0008537, referred to above.

FIG. 1 and FIG. 7 illustrate the measured DC response of a sampleapplied to the reagent chemistry for various tests where the timing ofthe application of the DC excitation signal after sample application isvaried from about 75 ms to 1400 ms. Hydration of FIG. 1 is less uniformstrip to strip than the thinner reagent in FIG. 7. Applying the DCexcitation too soon after sample application can result innon-Cottrellian responses and therefore inaccurate glucose concentrationmeasurements.

Using coating methods as generally described in U.S. Patent ApplicationPublication No. 2005/0008537, the reagent coating for FIG. 1 (50 g/m²coat weight) was approximately 3.6 μm and for FIG. 7 (20 g/m² coatweight) was approximately 1.6 μm. It can be seen that when the DCexcitation is applied very quickly after sample application to thethinner reagent layer, the response begins to exhibit the expectedCottrellian-like decay characteristics much more quickly, thereby makingaccurate determinations of the sample glucose concentration possible forfast test times. This is because enzyme availability, hydration, anddiffusion within the reagent layer, which limit how soon the DCmeasurement can be made, are improved with the thinner and or moreuniform reagent layer thickness. FIG. 30 shows a table of coat weightsettings and the estimated and actual measured dry coating filmthicknesses, using wet reagent coating methods described in the U.S.2005/0008537 publication. The equipment operating parameters required toachieve each coat weight will be appreciated from that publication andordinary skill in the art in that regard.

Technology and methodologies useful for forming thin reagent strips onbiosensors are disclosed in U.S. Patent Application Publication Nos.2005/0016844 and 2005/0008537, the disclosures of which are herebyincorporated by reference herein in their entireties.

FIG. 8 summarizes tests performed on biosensors having a reagent coatingformed thereon at thicknesses of 4 μm, 2.5 μm and 2.0 μm. Table 1 showsthe general formulation of the wet reagent coated on the biosensors usedin FIG. 8. The reagent was similar to that of ACCU-CHEK® AVIVA™biosensors, but prepared with a milled silica. Milled silica was used toreduce the mean particle size of the silica due to concerns that theunmilled silica may have particle sizes that would be detrimental tothinner coatings. They were coated at different coat weights leading todifferent measured thicknesses. The goal was to start with a reagentmass for glucose biosensors that had some previous optimization at leastfor the upper thickness level. Then a slot die coating method was usedto prepare reagent thicknesses from about 4 μm to 2 μm by adjusting thecoat weight using the same reagent mass. By making the reagents in thismanner, the concentrations of the active ingredients that were initiallyoptimized for the thicker coat weight are also reduced.

TABLE 1 Wet Reagent % w/w Keltrol F 0.22% CMC 0.57% Sipernat FK320 DS(milled) 2.02% PVP K25 1.91% Propiofan 2.88% GlucDOR wt 0.40% PQQ 0.01%Na-Succinate 0.29% Trehalose 0.48% KH₂PO₄ 0.39% K₂HPO₄ × 3H₂O 1.19%Mediator 31.1144 0.93% Mega 8 0.28% Geropon T77 0.03% KOH 0.14% Watertotal 88.27% Sum 100.00%

Blood samples were applied to each of the biosensors and AC excitationfrequencies of either 2 kHz or 20 kHz were applied to the biosensors asthe reagent was hydrated with the sample. Admittance data was measuredevery 100 ms for one second and plotted in FIG. 8. As can be seen, theAC admittance has stabilized in less than 400 ms after sampleapplication, and the AC data at 100 ms was shown to be adequate for usein correcting the resultant DC glucose test using the proceduredisclosed hereinbelow. From the data represented in FIGS. 1 and 7, it isclear that thin reagents stabilize quite fast after sample application.Of the films tested, the thinner reagents gave AC responses thatstabilized faster and in a more reproducible manner

The ability to achieve fast test times as disclosed herein is greatlyinfluenced by the rate of hydration of the enzyme and mediator in thereagent film, and the rate of diffusion of the reaction products to theelectrode surface under the reagent film. The use of the slot diecoating methodologies for deposition of reagent layers disclosed in U.S.Patent Application Publication Nos. 2005/0016844 and 2005/0008537 allowsdeposition of uniformly thin film reagents for faster and morereproducible dissolution of reagents, fill times and hydration profiles.FIG. 9 shows a surface profilometry measurement of a thin film reagentdeposited on a biosensor using these methods to a target thickness of2.5 μm (nominal coat weight=30 g/m²). As can be seen, the mean thicknessin the central B region of the reagent strip is 2.46 μm. Enzymeavailability, hydration, and diffusion are faster and more uniformlybehaved with thin reagent films (in one embodiment, approximately 1.6-10μm in thickness, and in other embodiments, approximately 1.6-5 μm inthickness).

Thin films benefit measurements in terms of faster hydration thatpermits measuring sooner after sample application. The AC stabilizationappears to be less affected by film thickness than the DC response. Amore Cottrellian-like behavior is observed in response to DC excitationat earlier times when the films are thinner. This can be seen bycomparison of FIG. 1 and FIG. 7. FIG. 1 shows current responses forthicker films, i.e. 50 g/m², which as illustrated give variable early Ivs. T traces when DC excitation is started around 100-700 ms aftersample sufficiency is detected. In contrast, as shown in FIG. 7, thecurrent responses follow a nice trend for the 20 g/m² for the same timerange. In addition, the I vs. T response becomes more Cottrellian-likeat about 300 ms after applying the DC potential. There are somelimitations that need to be considered with regard to thin films,however. There is a minimum amount of enzyme needed on the sensor bothfor obtaining a linear response and maintaining the required long-termstability of the sensor. The films in this example, because they weremade from the same reagent mass, had proportionally less enzyme as theywere made thinner. The lower limit of film thickness generally dependson the concentration of enzymes in the reagent mass to provide adequateresponse and stability. It is also understood that there would be somelower limit of thickness where the coating methods and variability ofthe thickness of the substrate would not provide a uniform coatingthickness. For other issues and disclosure relating to control ofuniform and homogeneous film thickness, see, e.g., U.S. PatentApplication Publication No. 2005/0008537 referred to hereinabove.

Example 2 Sequential Multiple AC Frequency Test with Fast Total TestTime and Varying Reagent Thickness

A covariate study testing multiple whole blood samples for glucoseconcentration was performed using the electrode and testing structuressimilar to the ACCU-CHEK® AVIVA™ biosensor available from RocheDiagnostics, Inc. of Indianapolis, Ind. USA. A pyrroloquinoline quinonedependent glucose dehydrogenase (PQQ-GDH) based reagent with the same orsubstantially similar formulation from Table 1 (above) was applied tothe biosensors in one of three thicknesses: 2 μm, 2.5 μm and 4 μm. Acovariate study was performed with whole blood samples similar toExample 1 but with six glucose concentrations, five hematocrit levels,and five temperatures, as detailed in FIG. 10.

The AC excitation potentials for this Example 2 were appliedsequentially as detailed in FIG. 11. A 10 kHz dose detection and samplesufficiency method (not shown) was followed by a 300 ms 20 kHz signalfollowed by 100 ms applications of 20 kHz, 10 kHz, 2 kHz and 1 kHzsignals. The measurement electrodes were then held at an open circuitfor 100 ms, followed by application of a 550 mV DC signal. Because ofthe pre-set timing parameters in the existing meters of the DATS, therewas a 50 ms stabilization delay and a 25 ms trailing data communicationperiod between each excitation block. Measurements of the response tothe DC signal were extracted at Total Test Times starting at about 1500ms, and measured at 100 ms intervals. AC admittance values were capturedfor each of the AC excitation blocks in order to correct the DC glucosemeasurement for the interfering effects of hematocrit and temperatureusing the following equation:

PredictedGlucose=INT+Yi2*Y2+Pi2*P2+Yi3*Y3+Pi3*P3+Yi4*Y4+Pi4*P4+Yi5*Y5+Pi5*P5+exp(SLOPE+Ys2*Y2+Ps2*P2+Ys3*Y3+Ps3*P3+Ys4*Y4+Ps4*P4+Ys5*Y5+Ps5*P5)*DC**POWER  (equation2)

where: Yi2, Yi3, Yi4, Yi5, Ys2, Ys3, Ys4 and Ys5 are constants

Pi2, Pi3, Pi4, Pi5, Ps2, Ps3, Ps4 and Ps5 are constants

Y2 is the admittance magnitude at 20 kHz (second block)

Y3 is the admittance magnitude at 10 kHz

Y4 is the admittance magnitude at 2 kHz

Y5 is the admittance magnitude at 1 kHz

P2 is the phase angle at 20 kHz (second block)

P3 is the phase angle at 10 kHz

P4 is the phase angle at 2 kHz

P5 is the phase angle at 1 kHz

INT is the intercept

SLOPE is the slope

DC is the uncorrected glucose response predicted with the DC measurement

POWER=Const+Yp2*Y2+Pp2*P2+Yp3*Y3+Pp3*P3+Yp4*Y4+Pp4*P4+Yp5*Y5+Pp5*P5

It will be appreciated that Equation 2 is substantially the same asEquation 1 from Example 1. The primary difference is only in thesequence order of applying the different frequencies, wherein theExample 1 applied frequency sequence was 10-20-2-1 kHz, and the Example2 applied frequency sequence was 20-10-2-1 kHz.

The uncorrected glucose response from the DC measurement (i.e.uncorrected for the interfering effects of hematocrit and temperature)was determined using well-known prior art techniques. This DC glucoseresponse was then corrected for the interfering effects of hematocritand temperature using the AC admittance magnitude and phase measurementdata as detailed above in equation 2. The Total System Error, bias,precision and NVar were calculated for each and these are tabulated inFIG. 12. As can be seen, the Total System Error for all three reagentthicknesses were very good at Total Test Times as low as 1.525 seconds.

As referred to above, Total System Error, or TSE, is a combined measureof accuracy and precision of the system. It is typically defined as:(Absolute Bias)+2*(Precision). The details are as follows:

Bias=Average of Normalized Error;

Precision=StdDev(Normalized Error);

where

Normalized Error=(Predicted Glucose−Reference Glucose) when ReferenceGlucose<=75 mg/dl; and

Normalized Error=(Predicted Glucose−Reference Glucose)*100/(ReferenceGlucose) when Reference Glucose>75 mg/dl.

FIG. 13 plots the normalized error versus the reference glucose valuefor the DC measurement data corrected using the AC measurements asdetailed hereinabove. Only the DC measurement taken at 1500 ms was used(+25 ms for communications), therefore this data represents a realistictotal test time of 1.525 seconds. The interfering effects of thehematocrit and temperature have been substantially reduced, with a TotalSystem Error of 10.0% for the entire covariate study.

FIG. 14 is a Clark Error Grid showing the predicted glucose value versusreference glucose value for all of the uncorrected DC glucosemeasurements taken at 1525 ms. The Clarke Error Grid Analysis (EGA) wasdeveloped in 1987 to quantify the clinical accuracy of patient estimatesof their current blood glucose as compared to the blood glucose valueobtained in their meter. See Clarke W L, Cox D, Gonder-Frederick L A,Carter W, Pohl S L: Evaluating clinical accuracy of systems forself-monitoring of blood glucose. Diabetes Care 10:622-628, 1987. TheClark Error Grid has since been used to quantify the clinical accuracyof blood glucose estimates generated by test meters as compared to areference value. The EGA is generally accepted as a standard methodologyfor determining the accuracy of blood glucose meters.

The Clark Error Grid breaks down a scatter plot of test results from areference glucose meter and an evaluated glucose meter into fiveregions. Region A are those values within 20% of the reference sensor,Region B contains points that are outside of 20% but would not lead toinappropriate treatment, Region C are those points leading tounnecessary treatment, Region D are those points indicating apotentially dangerous failure to detect hypoglycemia, and Region E arethose points that would confuse treatment of hypoglycemia forhyperglycemia and vice-versa. In FIG. 14, the dashed lines additionallyindicate values within 15% of the reference sensor.

As can be readily seen in FIG. 14, the uncorrected glucose values fallwell outside the +/−15% error window, which is the desired error windowset forth in a Clark Error Grid. This level of accuracy would beconsidered to be unacceptable in a glucose test meter according togeneral industry practice for blood glucose monitoring systems, as wellas according to FDA guidelines.

FIG. 15 is a Clark Error Grid showing the same DC test data shown inFIG. 14, except that the data has been corrected for the interferingeffects of hematocrit and temperature using the methodology describedhereinabove. As can be readily seen in FIG. 15, the performance of themeasurement system when corrected for hematocrit and temperature usingthe AC measurement data is far superior to using only the DC measurementresults to predict the glucose values at extremely fast Total TestTimes.

As can be seen from the above Example 2, the use of thin reagent films,such as about 1.6-5 μm in thickness supports the ability to performaccurate glucose determinations, corrected for the interfering effectsof hematocrit and temperature, with Total Test Times below 2 seconds.The uniformity of the reagent coating, and thus the resulting uniformdissolution/hydration of the reagent film with the fluid sample, isbelieved to enable reproducibility that correlates well with the ACmeasurements to provide accurately compensated glucose test results.

From Examples 1 and 2, it has become clear that, despite the previousunderstanding in the art, shorter test times can be achieved byshortened sequential AC blocks and/or by use of fewer sequential ACfrequencies. However, using more frequencies can provide benefits inmeasurement correction, especially when correcting for multiplevariables or when desired to actually provide an indication of the levelor general range of one or more such variables in addition to theanalyte measurement. In order to accomplish this, and still achieve theshortest possible test, the use of multi-frequency excitation waveformswas explored, such as set forth in Examples 3 and 4.

Multi-Frequency Excitation

As noted herein, some of the embodiments disclosed herein utilize thecollection of AC test data at multiple frequencies over a shorter timeperiod by using multi-frequency excitation waveform techniques. Thesemulti-frequency excitation waveforms are formed by adding a plurality ofindividual waveforms of varying frequency together so that the fluidsample is excited by multiple frequencies substantially simultaneously,rather than sequentially.

The resulting sample response can then be measured and this measurementwill contain the sample response to all of the excitation frequencies.The specific contribution from each excitation frequency component canbe then deduced by use of Fourier Transform techniques, such as aDiscrete Fourier Transform (DFT). Although the various examplesdisclosed herein utilize multi-sine excitation waveforms, those skilledin the art will recognize that the multi-frequency waveform may beconstructed using individual waveforms having any desired shape, such astriangular, square, sawtooth, delta, etc., just to name a fewnon-limiting examples. The component AC waveforms used to create themulti-frequency waveform may each have any desired frequency and anydesired amplitude. The use of multi-frequency techniques not onlyshortens the time necessary to collect the desired data (since the ACmeasurements are made simultaneously rather than sequentially), but alsocorrelates better for correction since the sample is varying less duringthe data collection corresponding to each applied frequency. This isparticularly true for tests utilizing a very fast Total Test Time, wherethe measurements are made very shortly after sample application and thesample is still undergoing diffusion and reaction with the reagentchemistry. Also, the AC measurement can be made closer in time to the DCmeasurement. Better correlation between the AC and DC allows for betterinterferent compensation even if the sample is not in steady state.

An exemplary prior art measurement sequence for a blood glucose testingsystem that corrects for the interfering effects of hematocrit andtemperature, such as those disclosed in U.S. Pat. No. 7,407,811, is asfollows:

-   Step 1: Blood is applied to a biosensor in a meter.-   Step 2: AC measurements are taken of the sample for drop detect    and/or dose sufficiency.-   Step 3: AC measurements are taken over a period of time to allow    calculation of correction factors for hematocrit and temperature. In    many instances, multiple AC excitation frequencies are applied to    the sample sequentially.-   Step 4: DC measurements are taken to measure the raw (uncorrected)    glucose response.-   Step 5: The raw DC response is compensated for hematocrit and    temperature effects using the AC measurement-derived correction    factors.-   Step 6: Measurement result is displayed to the user.

This procedure has some drawbacks with respect to obtaining ameasurement result in less than 2 seconds. While accurate measurementresults may be obtained by correcting the raw DC glucose measurementwith the AC-derived data on hematocrit and temperature, the additionaltime required to collect the AC data lengthens the total test time andalso separates in time the various AC and DC measurements that are usedto arrive at the final measurement result. This separation in time ofthe various AC and DC measurements can be of some concern in somesituations since the sample under test continues to undergo chemicalreactions with the reagents and the reagents are being hydrated duringthis time. That is, in a measurement sequence in which an AC signal isapplied with different waveform frequencies sequentially, the admittanceand phase data for each frequency, while still useful for the correctionof the subsequent raw DC response measurement, is not ideal because eachdata point is taken at a different time during the progression of thesample-reagent hydration-reaction dynamics. By applying all frequenciessimultaneously within the AC excitation waveform, the admittance andphase data for each frequency is still separately discernible andadvantageously relates to the same state of the sample-reagent dynamics.

A current response measured from application of an exemplarymulti-frequency AC excitation waveform followed by application of a DCsignal is illustrated in FIG. 29. For Examples 3 and 4, data acquisitionwas conducted using an electrochemical test stand constructed on thebasis of VXI components from Agilent, and programmable to apply AC andDC potentials to sensors in requested combinations and sequences and tomeasure the resulting current responses of the sensors.

Example 3 Multi-Frequency AC Test with Fast Total Test Time

The measurements conducted for Example 3 were made with electrodestructures similar to that of ACCU-CHEK® AVIVA™ biosensors and reagentsthe same or similar to the formulation set forth in Table 1 (above).These sensors were fabricated using generally the same technology asACCU-CHEK® AVIVA™ biosensors using a combination of processes includingsputtering, laser ablation, reagent slot die coating, and lamination.

The measurement sequence consisted of three basic blocks. The firstmeasurement block (not shown) utilized a 10240 Hz sine wave excitationapplied to the test strips in order to detect sample dose sufficiency(filling of the capillary test chamber sufficient to conduct ameasurement). The use of AC measurements for drop detect and dosesufficiency is described in U.S. Pat. No. 7,597,793, referred tohereinabove.

After sufficient sample was detected, the second measurement block wasbegun using a multi-sine (also known as polyphonic) waveform for a shorttime interval (as detailed below) to simultaneously collect ACadmittance magnitude and phase data for each frequency of interest. Themulti-sine waveform used for this Example 3 was constructed by summingsine waves of four frequencies (1024, 2048, 10240 and 20480 Hz). Thesefrequencies were selected because they are known to be useful forcorrection of interferents, according to Applicants' prior disclosuresregarding use of AC excitation which are referred to hereinabove. Thehigher frequency ranges of about 20 and about 10 kHz are known toprovide useful correction for hematocrit. The lower frequency ranges ofabout 1 and about 2 kHz were included because of the known potential foruseful discrete measurements. Generally, this combination of frequenciesallow for correction of multiple parameters such as hematocrit andtemperature. It is well understood that these values do not have to bespecifically 20 kHz, for example, but only in a range where theinterferents can be measured reasonably independent of the glucoseresponse which is to be corrected. A higher frequency may correlate morewith one interferent such as hematocrit, whereas another frequency maycorrelate more with another interferent. Optimization of the frequencyor combination of frequencies that would provide the best overallcorrection response would be useful, and is well within the skill of aperson of ordinary skill in the art in view of this disclosure. However,in working with multi-frequency AC waveforms to reduce the time tocollect response data from multiple frequencies while still providinggood correction and short total test times, it was decided that usingthe frequencies in these known ranges would be useful in order to relyon past experience. In addition, previous experience shows that datafrom more than one frequency can correct for multiple interferentsbetter than measuring at only one frequency. Four frequencies werechosen here so that previously programmed data analysis routines couldbe used. However, two, three or even five or more frequencies, forexample, may just as well supply adequate correction. Some discrete ACmethods with only two AC frequencies have been conducted.

For Example 3, the multi-sine waveform consisted of one period of the1024 Hz signal, two periods of the 2048 Hz signal, 10 periods of the10240 Hz signal, and 20 periods of the 20480 Hz signal. The peakamplitude was set at 12.7 mV, but due to the multi-sine nature of thesignal, the actual RMS value would be significantly lower. (RMS is theroot mean square value of the waveform SQRT[(1/N)*SUM(x²)].) Thewaveform comprised 16000 data points that were input to adigital-to-analog converter and is illustrated in FIG. 16.

One benefit of using the multi-sine excitation waveform is that the ACmeasurement time required to collect data for all four frequencies isreduced because the measurements are made simultaneously. Anotherbenefit of the multi-sine excitation waveform is that the AC measurementdata for all of the frequencies is collected simultaneously and is thusless affected by the fact that the sample is changing as it reacts withthe reagent.

The multi-sine waveform was applied to the test sample for 300 ms afteran indication of dose sufficiency and analyzed in 100 ms intervals.Although this Example 3 utilized a 300 ms measurement period, longer,shorter and even variable time periods may be employed with similarresults. Generally, to achieve a Total Test Time of two seconds or less,the range for the multi-sine measurement period in one embodiment is 100ms to 1900 ms. With the ACCU-CHEK® AVIVA™ test structures used, 200-500ms was a sufficient period to give reproducible AC responses from thetest sample.

Although the various excitation frequencies are applied to the samplesimultaneously using the multi-sine signal, the responses attributableto each frequency component can be extracted from the AC measurementdata using an appropriate mathematical function, such as a Fast FourierTransform (FFT) or a Discrete Fourier Transform (DFT) or othermathematical techniques, as will be appreciated by those skilled in theart. Admittance magnitude and phase data for each frequency wasextracted in the present Example 3 using DFT. This extracted admittancedata for each frequency is shown in FIG. 17A for the time pointcorrelating to 200 ms after dose sufficiency for all nine samplestested. A graph of phase to hematocrit at each frequency for Example 3is illustrated in FIG. 17B, and a graph of admittance magnitude tohematocrit at each frequency for Example 3 is illustrated in FIG. 17C.

The second measurement block consisted of a 550 mV DC signal applied tothe sample in order to obtain a raw (uncorrected) predicted glucosereading, as is known in the art. Four DC time points were extracted fromthe measurement data as 100 ms average data points with ending datapoints at 500, 600, 1000 and 1500 ms (i.e. for Total Test Times of 0.5,0.6, 1.0 and 1.5 seconds).

Nine whole blood samples were prepared for a covariate study usingtarget glucose concentrations of 90, 250 and 600 mg/dL and targethematocrit values of 20, 45 and 70%. For each sample tested, each DCtime point was analyzed by nonlinear fit and the 300 ms AC admittancemagnitude and phase data was used to calculate the predicted glucoseresponse compensated for the effects of hematocrit and temperature usingthe following equation:

PredictedGlucose=INT+Yi1*Y1+Pi1*P1+Yi2*Y2+Pi2*P2+Yi3*Y3+Pi3*P3+Yi4*Y4+Pi4*P4+exp(SLOPE+Ys1*Y1+Ps1*P1+Ys2*Y2+Ps2*P2+Ys3*Y3+Ps3*P3+Ys4*Y4+Ps4*P4)*DC**POWER  (equation3)

where: Yi1, Yi2, Yi3, Yi4, Ys1, Ys2, Ys3 and Ys4 are constants

Pi1, Pi2, Pi3, Pi4, Ps1, Ps2, Ps3 and Ps4 are constants

Y1 is the admittance magnitude at 1024 Hz

Y2 is the admittance magnitude at 2048 Hz

Y3 is the admittance magnitude at 10240 Hz

Y4 is the admittance magnitude at 20480 Hz

P1 is the phase angle at 1024 Hz

P2 is the phase angle at 2048 Hz

P3 is the phase angle at 10240 Hz

P4 is the phase angle at 20480 Hz

INT is the intercept

SLOPE is the slope

DC is the uncorrected glucose response predicted with the DC measurement

POWER is=Const+Yp1*Y1+Pp1*P1+Yp2*Y2+Pp2*P2+Yp3*Y3+Pp3*P3+Yp4*Y4+Pp4*P4.

Again this equation is the same form as Equations 1 and 2, but as can beseen the variables range from Y1 to Y4 and P1 to P4 for the simultaneousAC at 200 ms, rather than Y2-Y5 and P2-P5 which are used in Equations 1and 2.

As discussed above, the use of AC admittance magnitude and phase data tocorrect DC glucose response data for the effects of hematocrit andtemperature is discussed in U.S. Pat. No. 7,407,811.

The uncorrected glucose response from the DC measurement (i.e.uncorrected for the interfering effects of hematocrit and temperature)was determined using well-known prior art techniques. This DC glucoseresponse was then corrected for the interfering effects of hematocritand temperature using the AC admittance magnitude and phase measurementdata as detailed above in equation 3. The Total System Error (TSE),bias, precision and NVar were calculated for each Total Test Time (forboth corrected and uncorrected results) and these are tabulated in FIG.18. As can be readily seen, the performance of the measurement systemwhen corrected for hematocrit and temperature using the AC measurementdata is far superior to using only the DC measurement results to predictthe glucose values. Furthermore, acquiring the AC measurement datasimultaneously for multiple excitation frequencies permits extremelyfast Total Test Times with measurement results exhibiting very good TSEvalues for Total Test Times of 1.5 seconds, 1.0 second, 0.6 seconds and0.5 seconds.

FIG. 19 plots the normalized error versus the reference glucose valuefor the uncorrected glucose measurements, and a significant dependenceon the hematocrit value can be seen in the data. The Total System Erroris 53.8%. FIG. 20 plots the normalized error versus the referenceglucose value for the same measurements, only this time the DCmeasurement data has been corrected using the AC measurement as detailedhereinabove. The interfering effect of the hematocrit has clearly beensubstantially reduced, with a Total System Error of 14.2%. Thisreduction was achieved using only the AC measurement data taken at 500ms after the dose sufficiency indication.

FIG. 21 is a Clark Error Grid showing the predicted glucose value versusreference glucose value for all of the above 500 ms data points, bothcorrected and uncorrected. As can be readily seen, the uncorrectedglucose values fall well outside the +/−15% error window, while thecorrected data are all within this limit. Therefore, the use ofmulti-frequency excitation to achieve a one-half second glucose TotalTest Time was demonstrated.

The above data of this Example 3 clearly shows that the use of themulti-frequency excitation techniques disclosed herein allow extremelyshort test times by allowing the sample to be simultaneously excited bymultiple frequencies and the sample responses to those frequencies to besimultaneously measured. Even at one-half second Total Test Times, thedata provides a significant reduction in the DC measurement error causedby interferents, and allows essentially instantaneous measurementresults to be reported to the user with a measurement accuracy wellwithin accepted industry standards.

Example 4 Multi-Frequency AC Test with Fast Total Test Time

The measurements conducted for Example 4 were made with the sameelectrode structures and reagents as Example 3 and the same measurementsequencing. However, whereas the Example 3 measurements were performedon samples having three different target analyte concentrations andthree different hematocrit levels for each concentration, the Example 4measurements were performed on samples having seven different targetanalyte concentrations and three hematocrit levels for eachconcentration.

As was found in Example 3, it was learned from the measurements ofExample 4 that a benefit of using the multi-sine excitation waveform isthat the AC measurement time required to collect data for all fourfrequencies is reduced because the measurements are made simultaneously.Another benefit of the multi-sine excitation waveform is that the ACmeasurement data for all of the frequencies is collected simultaneouslyand is thus less affected by the fact that the sample is changing as itreacts with the reagent.

Twenty-one whole blood samples were prepared for a covariate study usingtarget glucose concentrations of 50, 100, 140, 250, 300, 450 and 550mg/dL and target hematocrit values of 20, 45 and 70%. For each sampletested, each DC time point was analyzed by nonlinear fit and the 300 msAC admittance magnitude and phase data was used to calculate thepredicted glucose response compensated for the effects of hematocrit andtemperature using the following equation:

PredictedGlucose=INT+Yi1*Y1+Pi1*P1+Yi2*Y2+Pi2*P2+Yi3*Y3+Pi3*P3+Yi4*Y4+Pi4*P4+exp(SLOPE+Ys1*Y1+Ps1*P1+Ys2*Y2+Ps2*P2+Ys3*Y3+Ps3*P3+Ys4*Y4+Ps4*P4)*DC**POWER  (equation4)

where: Yi1, Yi2, Yi3, Yi4, Ys1, Ys2, Ys3 and Ys4 are constants

Pi1, Pi2, Pi3, Pi4, Ps1, Ps2, Ps3 and Ps4 are constants

Y1 is the admittance magnitude at 1024 Hz

Y2 is the admittance magnitude at 2048 Hz

Y3 is the admittance magnitude at 10240 Hz

Y4 is the admittance magnitude at 20480 Hz

P1 is the phase angle at 1024 Hz

P2 is the phase angle at 2048 Hz

P3 is the phase angle at 10240 Hz

P4 is the phase angle at 20480 Hz

INT is the intercept

SLOPE is the slope

DC is the uncorrected glucose response predicted with the DC measurement

POWER is=Const+Yp1*Y1+Pp1*P1+Yp2*Y2+Pp2*P2+Yp3*Y3+Pp3*P3+Yp4*Y4+Pp4*P4.

Again, Equation 4 is the same form as Equations 1 and 2, but as withEquation 3 from Example 3, one can see that the variables range from Y1to Y4 and P1 to P4 for the simultaneous AC at 200 ms, rather than Y2-Y5and P2-P5 which are used in Equations 1 and 2.

As discussed in Example 3, the use of AC admittance magnitude and phasedata to correct DC glucose response data for the effects of hematocritand temperature is discussed in U.S. Pat. No. 7,407,811. A graph ofadmittance magnitude to hematocrit at each frequency for Example 4 isillustrated in FIG. 27, and a graph of phase to hematocrit at eachfrequency for Example 4 is illustrated in FIG. 28.

The uncorrected glucose response from the DC measurement (i.e.uncorrected for the interfering effects of hematocrit and temperature)was determined using well-known prior art techniques. This DC glucoseresponse was then corrected for the interfering effects of hematocritand temperature using the AC admittance magnitude and phase measurementdata as detailed above in equation 4. The Total System Error (TSE),bias, precision and NVar were calculated for each Total Test Time (forboth corrected and uncorrected results) and these are tabulated in FIG.22. As can be readily seen, the performance of the measurement systemwhen corrected for hematocrit and temperature using the AC measurementdata is far superior to using only the DC measurement results to predictthe glucose values. Furthermore, acquiring the AC measurement datasimultaneously for multiple excitation frequencies permits extremelyfast Total Test Times. As shown in FIG. 22, measurement results exhibitvery good TSE values at Total Test Times of 1.525 seconds, 1.025seconds, 0.725 seconds and 0.625 seconds.

FIG. 23 plots the normalized error versus the reference glucose valuefor the uncorrected glucose measurements, and a significant dependenceon the hematocrit value can be seen in the data. The Total System Erroris 47.5%. FIG. 24 plots the normalized error versus the referenceglucose value for the same measurements, only this time the DCmeasurement data has been corrected using the AC measurement as detailedhereinabove. The interfering effect of the hematocrit has clearly beensubstantially reduced, with a Total System Error of 10.2%. Themeasurement data for each of the 21 measurement runs can be seen in FIG.26.

FIG. 25 is a Clark Error Grid showing the predicted glucose value versusreference glucose value for all of the 725 ms data points, bothcorrected and uncorrected. As can be readily seen, most of theuncorrected glucose values fall well outside the +/−15% error window,while the corrected data are all within this limit. Therefore, the useof multi-frequency excitation to achieve a less than three-quarters of asecond glucose Total Test Time was demonstrated.

The above data of this Example 4 clearly shows that the use of themulti-frequency excitation techniques disclosed herein allow extremelyshort test times by allowing the sample to be simultaneously excited bymultiple frequencies and the sample responses to those frequencies to besimultaneously measured. Even at sub-three-quarter second Total TestTimes, the data provides a significant reduction in the DC measurementerror caused by interferents, and allows essentially instantaneousmeasurement results to be reported to the user with a measurementaccuracy well within accepted industry standards.

Example 5 Sequential Multiple AC Frequency Test with Fast Total TestTime at Various DC Time Points

This Example 5 was conducted similarly to Example 2 (above), using testsensors having a reagent film thickness based on a nominal 30 g/m² coatweight application which, as shown in FIG. 30, corresponds approximatelyto a thickness of 2.45 μm. Unlike Example 2, however, data acquisitionwas conducted using an electrochemical test stand constructed on thebasis of VXI components from Agilent, and programmable to apply AC andDC potentials to sensors in requested combinations and sequences and tomeasure the resulting current responses of the sensors. This was donebecause, as noted with regard to Examples 1 and 2, the existing metersused with the DATS for those measurements comprise pre-set parameters inwhich mandatory time blocks are required for a preceding waveformstabilization, trailing communication after each frequency block, and apre-set “skip” period in the initial 100 ms of DC signal applicationduring which no current response can be measured. For this Example 5,however, it was desired to apply sequential multiple AC frequencieswithout the limitations of the pre-set timing conditions imposed by theexisting meters of the DATS. The Agilent-based test stand used forExample 3 and 4 provided the flexibility to program a desiredmeasurement sequence in this way.

The purpose of Example 5 was to explore different ways in which a set offour short 200 ms AC excitation blocks applied sequentially and havingfrequencies of 20, 2, 10, and 1 kHz can be used to correct a DC glucoseresponse, co-varied with hematocrit, at a single generally uniformreagent film thickness. The AC excitation blocks were applied startingat a time zero, which is the time at which sufficient sample dosing isdetected. Thus, the AC excitation blocks begin at time zero with no openperiod in between, ending at about 800 ms, at which time the DCexcitation was applied.

The DC response data was collected starting at 800 ms through 3700 ms.This dataset was used to analyze the data with varying AC and DCparameters. The goal was to determine if good performance could bereached with this uniform thin film at short test times, and todetermine the effect of using one or multiple AC responses for thecorrections. FIG. 31 shows the AC response vs. hematocrit for the 4 ACfrequencies measured. All of these data show an inverse relationship ofadmittance response to increased hematocrit. As can be seen, themeasurements at the higher frequencies of 20 kHz and 10 kHz exhibitgenerally similar responses, and the measurements at the lowerfrequencies of 2 kHz and 1 kHz exhibit generally similar responses.However, the higher frequencies have a greater hematocrit vs. admittancerelationship.

FIG. 32 shows uncorrected DC response data collected for this covariatestudy and it is clear that at each DC test time there are variablecurrent responses associated with the varied hematocrit levels. FIG. 33shows a typical response of an uncorrected Normalized Error to areferenced glucose value for the covariant study using the DC responsemeasured at 900 ms. The uncorrected response has a TSE of 41%. However,as shown in FIG. 34, when the DC response at 900 ms is corrected usingthe AC response data from just two frequencies, namely 20 kHz and 2 kHz,there is significant improvement of the TSE, which has decreased to7.1%. FIGS. 35-39 show Normalized Error plots for the corrected DCresponse measured at 1100 ms, 1500 ms, 2000 ms, 2500 ms, and 3000 ms,respectively, again corrected using only the AC response data from the20 kHz and 2 kHz sequentially applied frequencies of the AC signal.

FIG. 40 shows a table of the TSE according to variable DC test time forDC response data corrected in two ways, first with the 20 kHz and 2 kHzAC response data and second with the 10 kHz and 1 kHz AC response data.The AC response data at 20 kHz and 2 kHz provided a better correctionfor this test configuration (in terms of TSE) than the 10 kHz and 1 kHzresponse data.

FIG. 40 also indicates that at a short test time of 900 ms the TSE isactually better than at longer times; that is, there is an increase inTSE as the DC measurement time increases, but this is then followed by adecrease in TSE at the much longer time of 3000 ms. It is believed thatthe TSE is lower at the shorter DC measurement times because at theshorter DC measurement times the time between measurement of the ACresponses (for obtaining the correction factors) and the measurement ofthe DC response (for obtaining analyte-related data) is on the order ofonly 100-900 ms. That is, the AC response data is obtained approximatelyat times 200 ms, 400 ms, 600 ms and 800 ms, with the shorter DC responsedata being obtained at 900 and 1100 ms. Thus, the correction factorresponses and the analyte response correlate best when film hydrationand reaction properties are almost identical. At shorter DC responsemeasurement times measurements are made closer to the electrode surfacewith short diffusion distances where there is less effect due to filmhydration and swelling.

As measurements are made at moderately longer DC response times, the TSEincreases because the AC correction factors and the DC response arefurther apart (less correlated) because the film is hydrating andswelling rapidly and the DC response is being measured in this region ofrapid change. However at even longer DC measurement times, e.g. 3000 ms,the TSE comes back down when the reagent hydration and swelling beginsto stabilize, causing the DC value to have less variability and needingless correction by the AC correction factors. Thus, at these longermeasurement times, the TSE appears to improve to values close to the TSEof the earlier DC response measurement time. Typically, AC/DC responsestaught in prior art disclosures measured the DC response data when theDC response was most stable, which is typically later, and thus lostsome of the correlation between correction factors and analyte response.Here we show that we can measure the DC response at earlier measurementtimes and still obtain an acceptable analyte response with the addedbenefit of reduced test time. In the case of this Example 5, the TotalTest Time is less than 1 second (i.e. 900 ms).

It is also believed that the AC correction factors disclosed hereincorrect not just the hematocrit affects but also other sources of erroror variability of the reagent film state. In the examples describedherein, the electrodes used to detect the AC signal response are thesame ones used for the DC signal response, and thus are coated with thereagent film. As a result, all AC response measurements are affected bythe state of the reagent film with liquid sample applied (e.g.thickness, swelling).

Another way to look at these data is from corresponding Clark ErrorGrids. FIG. 41 shows the Error Grid for the uncorrected DC response dataat a 900 ms DC measurement time. FIGS. 42-44 show the same 900 ms DCmeasurement data corrected with AC response data for only 20 kHz (FIG.42), only 2 kHz (FIG. 43), and both 20 kHz and 2 kHz (FIG. 44).

The data from Example 5 supports a finding that an analyte measurementhaving good TSE can be achieved at short Total Test Times, between 900ms and 3000 ms.

Example 5 was not co-varied with temperature as was done in Example 2because the electrochemical test stand was less conducive to running“environmental” studies. Thus, the AC signal responses or correctionfactors determined from those responses in this example do not containinformation on sample temperature variations and correction as was shownin Example 2. However, AC methods using the 4 AC frequencies have beenshown to correct both hematocrit and temperature variations, and themeasurement method of Example 5 would be sufficient to do this with atest time of less than 1000 ms.

For purposes of the examples disclosed herein, the DC excitation appliedis described and shown generally as a single block of applied potentialfor a single duration. DC response data may be taken throughout thatduration or only at one or only a few points in that duration. Not shownor described, however, is a DC excitation application which comprisestwo or more shorter pulses of DC excitation with response data measuredfor each such pulse. While none of the examples herein illustrate theuse of this type of DC excitation, it is believed that the AC waveformsdescribed herein, both sequential and multi-frequency (simultaneous)waveforms, can correct the response data obtained from the pulsed typeof DC excitation.

The features disclosed in the above description, the claims and thedrawings may be important both individually and in any combination withone another for implementing the invention in its various embodiments.

It is noted that terms like “preferably”, “commonly”, and “typically”are not utilized herein to limit the scope of the claimed invention orto imply that certain features are critical, essential, or evenimportant to the structure or function of the claimed invention. Rather,these terms are merely intended to highlight alternative or additionalfeatures that may or may not be utilized in a particular embodiment ofthe present invention.

For the purposes of describing and defining the present invention it isnoted that the term “substantially” is utilized herein to represent theinherent degree of uncertainty that may be attributed to anyquantitative comparison, value, measurement, or other representation.The term “substantially” is also utilized herein to represent the degreeby which a quantitative representation may vary from a stated referencewithout resulting in a change in the basic function of the subjectmatter at issue.

Having described the present invention in detail and by reference tospecific embodiments thereof, it will be apparent that modification andvariations are possible without departing from the scope of the presentinvention defined in the appended claims. More specifically, althoughsome aspects of the present invention are identified herein as preferredor particularly advantageous, it is contemplated that the presentinvention is not necessarily limited to these preferred aspects of thepresent invention.

1. A method for determining a concentration of an analyte of interest ina biological sample, comprising: a) applying a first signal having afirst AC component to the sample; b) measuring a first response to thefirst signal; c) applying a second signal having a second AC componentto the sample; d) measuring a second response to the second signal; e)applying a third signal having a third AC component to the sample; f)measuring a third response to the third signal; g) applying a fourth DCsignal to the sample; h) measuring a fourth response to the fourth DCsignal; and e) determining the analyte concentration of the sample usingat least the first, second and third responses to correct the fourthresponse for the effects of at least one interferent within the sample,wherein the determination has a Total Test Time of within about 2.0seconds or less, and wherein the first, second third, and fourth signalsare applied to the sample during separate time periods.
 2. The method ofclaim 1, wherein the determination has a Total Test Time of betweenabout 0.5 seconds and about 1.525 seconds.
 3. The method of claim 1,wherein the method has a Total System Error of less than about 11%. 4.The method of claim 1, wherein the biological sample is blood and theanalyte of interest is glucose.
 5. The method of claim 1, wherein thefirst, second and third signals each comprise an AC signal, each ACsignal having a different frequency, and wherein the fourth signalcomprises a DC signal.
 6. The method of claim 1, wherein the interferentcomprises one or both of hematocrit and temperature.
 7. The method ofclaim 1, wherein the first signal is applied for no more than about 100ms before the first response is measured; wherein the second signal isapplied for no more than about 100 ms before the second response ismeasured; and wherein the third signal is applied for no more than about100 ms before the third response is measured.
 8. The method of claim 1,wherein the fourth signal is applied for no more than about 200 msbefore the fourth response is measured.
 9. The method of claim 1,further comprising: applying a fifth signal having a fourth AC componentduring a separate time period from the first, second, third and fourthsignals; and measuring a fifth response to the fifth signal; whereinsaid determination of the analyte concentration of the sample uses atleast the first, second, third and fifth responses to correct the fourthresponse for the effects of at least one interferent within the sample.10. The method of claim 9, wherein the first, second, third and fifthsignals each comprise an AC signal, each AC signal having a differentfrequency, and wherein the fourth signal comprises a DC signal.
 11. Themethod of claim 9, wherein the first signal is applied for no more thanabout 100 ms before the first response is measured; wherein the secondsignal is applied for no more than about 100 ms before the secondresponse is measured; wherein the third signal is applied for no morethan about 100 ms before the third response is measured; and wherein thefifth signal is applied for no more than about 100 ms before the fifthresponse is measured.
 12. The method of claim 9, wherein the firstsignal is a 10 kHz AC signal, the second signal is a 20 kHz AC signal,the third signal is a 1 kHz AC signal, and the fifth signal is a 2 kHzAC signal.
 13. The method of claim 9, wherein the fourth signal isapplied for no more than about 200 ms before the fourth response ismeasured.
 14. The method of claim 9, wherein the method has a TotalSystem Error of less than about 11%.
 15. The method of claim 14, whereinthe method has a Total System Error of between about 7.5% and about 11%.